Pain is perceived as a result of communication between the two main divisions—central and peripheral—of the nervous system. While the two divisions work together to produce our subjective experience, the central and peripheral nervous systems are anatomically and functionally different.
A painful stimulus impinging on a specialized pain receptor is propagated along a peripheral branch of a primary nociceptive sensory neuron whose cell body resides within a dorsal root ganglion (part of the peripheral nervous system) and then along a central branch of the neuron that enters the spinal cord (central nervous system). The signal is subsequently relayed to a second order neuron in the spinal cord that, in turn, transmits the signal to the opposite (“contralateral”) side of the spinal cord. The signal is then communicated to higher centers in the brain where it is perceived as painful.
Peripheral pain receptors, which respond to mechanical, thermal or chemical stimuli are located on nerve endings of the primary nociceptive neurons. Activation of these receptors results in pain that can be acute or chronic. Acute pain tends to be sharp and well-localized and is typically transmitted along the thinly myelinated axons of A delta sensory neurons. Chronic pain is usually dull and diffuse, and is conveyed along non-myelinated axons of C-type nociceptive neurons. Chemical mediators of inflammation such as bradykinin and prostaglandins stimulate pain receptors, and are important agents in chronic pain syndromes, such as the persistent pain associated with arthritis, ileitis or cystitis, to name but a few.
The perception of pain can be altered at various stages of the pain pathway. For example, administering a local anesthetic to the peripheral receptor can eliminate the painful stimulus. Drugs like opioids were classically known to intervene at the central nervous system stage of the pain pathway, and non-steroidal anti-inflammatory drugs at the peripheral stage (although it is now realized that there is some cross-reactivity of both). Likewise, what is perceived as chronic pain (not due to primary spinal cord injury) is typically associated with sensitization of peripheral pain receptors as well as changes in the excitability of the second order neurons, and therefore has both peripheral and central nervous system components. The peripheral and central components regulate “primary” and “secondary” hyperalgesia, respectively (Urban and Gebhart, 1999, citing Woolf, 1983 and La Motte et al., 1991). In secondary hyperalgesia, the second order neuron in the central nervous system undergoes changes in gene expression that contribute to the phenomenon of “central sensitization” or “spinal hyperalgesia”. Spinal N-methyl-D-aspartate (“NMDA”) receptors are believed to play an important role in this process (Urban and Gebhart, 1999, citing Urban and Gebhart, 1998; Palacek et al., 2003; Lee et al., 1993). Spinal cord injury (presumably) without activation of the peripheral nervous system can also produce spinal hyperalgesia resulting in a central pain syndrome (Zhang et al., 2005). Central neuropathic pain has been associated with phosphorylation of the transcription factor, cyclic AMP response element binding protein (“CREB”) (Cron et al., 2005).
Chronic pain is initiated in the periphery by either a nerve injury (“neuropathic pain”) or an inflammation and both sources result in pain that is a major clinical problem that has mostly resisted effective treatment. In humans (Gracely et al., 1992) and mammalian model systems (Millan, 1999), persistent pain after nerve injury is associated with long-term hyperexcitability (LTH) of those primary sensory neurons whose axons are in the affected nerve. LTH is manifested as increased sensitivity to electrical stimuli in the nociceptive sensory neuron cell body and axon at the injury site (Wall and Devor, 1983; Study and Kral, 1996; Zhang et al., 1997; Chen and Devor, 1998; Kim et al., 1998; Abdulla and Smith, 2001). These changes result in the discharge of action potentials from sensory neurons at rest or during innocuous stimulation, leading to continuing excitation of higher order neurons in the central nervous system, spinal hyperalgesia and persistent pain. Because the appearance of LTH involves alterations in gene expression (Waxman et al., 1994; Wang et al., 2002; Park et al., 2003), a central question is, how are such changes in the neuron nucleus induced by an injury that occurs far from the cell body? Answering this question has been extremely difficult using the complex mammalian nervous system.
An experimentally favorable alternative is the homogeneous cluster of nociceptive sensory neurons that reside in the bilateral pleural ganglia of the mollusk Aplysia californica (Walters et al., 2004). Noxious mechanical stimulation of the body wall (Walters et al., 1983a) or crushing sensory neuron axons in vivo or in vitro elicits an LTH with electrophysiological properties similar to those seen after axotomy of mammalian nociceptive neurons (Walters et al., 1991; Walters, 1994; Ambron et al., 1996; Bedi et al., 1998; Ungless et al., 2002; Sung and Ambron, 2004). The LTH appears after a delay, suggesting that its induction after nerve crush is attributable to a positive molecular injury signal (Walters et al., 1991; Ambron and Walters, 1996; Lin et al., 2003). Two studies support this idea. First, blocking axonal transport after nerve injury in excised nervous systems prevented the appearance of LTH (Gunstream et al., 1995). Second, LTH was induced in noninjured sensory neurons by injecting axoplasm from injured axons (Ambron et al., 1995). LTH was also elicited in the neurons after intrasomatic injection of an ERK (extracellular signal-regulated kinase) member of the MAPK (mitogen-activated protein kinase) family (Sung et al., 2001). Other experiments have suggested that cyclic GMP (cGMP) and PKG (cGMP-dependent protein kinase; protein kinase G) are probably involved (Lewin and Walters, 1999). However, despite these observations, it was only recently that the signal from the axon was identified.
U.S. Pat. No. 6,476,007 by Tao and Johns (“the '007 patent”) relates to a proposed signalling pathway in the central nervous system in which stimulation of an N-methyl-D-aspartate (“NMDA”) receptor leads to activation of nitric oxide synthase (“NOS”) and production of nitric oxide (“NO”), which then stimulates guanylate cylase (“GC”) and the production of cyclic guanoside monophosphate (cGMP), which in turn activates cGMP-dependent protein kinase I (“PKG”). It was observed that administration of the PKG inhibitor Rp-8-[4-chlorophenyl)thio]-cGMPS triethylamine into the central nervous system by intrathecal administration, after the induction of an inflammatory response, produced significant attenuation of acute pain in rats 10 and 60 minutes later. Further, the inventors of the '007 patent noted an upregulation of PKG expression in the lumbar spinal cord 96 hours after noxious stimulation was blocked by administration of a neuronal NOS inhibitor, a soluble GC inhibitor, and a NMDA receptor antagonist.
However, the '007 patent is directed toward the mechanism of inflammatory hyperalgesia in the central nervous system; the role of the peripheral nervous system is not considered. Targeting the pain pathway in the central nervous system suffers from several important disadvantages. First the neuronal circuits in the spinal cord are highly complex and not well understood. Thus, drugs that might be predicted to relieve pain can have the opposite effect. Second, the neurons in the central nervous system are sequestered from the rest of the body by the blood-brain-barrier, which is a formidable obstacle that often prevents many therapeutic drugs from ever reaching their targets. The limited permeability means that treatment of spinal hyperalgesia according to the '007 patent would be problematic. Third, drugs that do penetrate the blood brain barrier have access to the entire central nervous system so that side effects can be severe. In contrast, there is no such barrier in the peripheral nervous system. Moreover, the anatomical disposition of the DRG means that it is possible to target specific populations of primary sensory neurons for treatment. Fourth, pain as a sensation is perceived only when signals from the periphery are communicated to higher centers in the brain. Consequently, since the DRG neurons are the portal for these signals, the present invention offers the advantage of intervening in subjective pain as it first arises. Finally, the 007 patent describes methods to prevent the activation of PKG; it does not address the inhibition of the already activated PKG.
Active PKG has a critical role in the initiation of pain. (See International Patent Application No. PCT/US2006/010107, Publication No. WO2006/102267). Following injury to a peripheral nerve there is an increase in nitric oxide synthase (“NOS”) activity that results in increased nitric oxide (“NO”) production. The NO activates soluble guanylyl cyclase (“sGC”), thereby increasing levels of cyclic guanosine monophosphate (“cGMP”) which results in the activation of protein kinase G (“PKG”) in the axons of the C-type and A-delta type nociceptive neurons. The activated PKG is then retrogradely transported from the site of injury along the axon to the neuron cell body, where it phosphorylates mitogen-activated protein kinase-erk (“MAPKerk”) (Sung et al., Aug. 25, 2004). The activated MAPKerk then translocates into the cell nucleus, where it modulates expression of the pain-related genes that mediate the appearance of LTH. Since inhibiting PKG attenuates pain and reduces the level of mRNAs for proteins that are involved in nociception, the focus of the present invention relates to modulators of the activated PKG.
Balanol is a known protein kinase C (PKC) inhibitor. Various balanol analogs which inhibit PKC have been previously identified by a retro-synthesis of balanol isolated from Verticillium balanoides (Lai et al. 1997). The retro-synthesis of the compound divided the compound into the following three main constituents: a tetrasubstituted benzophenone diacid, a trans-3,4-aminohydroxyperhydroazepine, and a 4-hydroxybenzoic acid. The balanol analogs were then synthesized with replacement of the perhydroazepine moiety. Specifically, Lai compared the activity of the analogs to balanol, the parent compound, and found that the analogs were more isozyme selective, demonstrating more selectivity between PKC and PKA than the parent compound (Lai et al. 1997). Lai concluded that the activity and the selectivity of the compounds was largely related to the conformation of the nonaromatic structural elements of the molecule. Ring size of the pyrrolidine nitrogen was found to greatly affect potency, with five molecules considered to have optimal potency.
While Lai was directed to analog development, the focus on the pyrrolidine ring, while valuable in its findings, is limited. The value of different or additional varying substituents at other ring sites within the compound, and the advantage of PKG selective inhibitory activity, were not considered prior to the present invention.
The prior art has demonstrated some additional compounds that exhibit PKC inhibitory action. For example, U.S. Pat. No. 5,432,198 by Jadgdmann et al. (“the '198 patent) discloses additional balanol analogues with different substituents, wherein the compounds have PKC inhibitory activity. The '198 patent discloses a balanol analogue without a pyrrolidine nitrogen, but instead has a carbon ring up to 7 members. Among other substitutions, the '198 compound also requires an alkyl substituted aromatic ring on the amine end of the compound.
U.S. Pat. No. 5,583,221 by Hu et al. (“the '221 patent”) similarly discloses compounds that exhibit PKC inhibitory activity. However, the '221 patent is limited in that it does not cover balanol derivatives or pyrrolidine-containing compounds. U.S. Pat. Nos. 6,376,467 and 6,686,334 by Messing et al. (“the '467 patent” and “the '334 patent”, respectively) disclose methods to lessen pain with compounds that are specifically directed to an inhibitor of the e isozyme of PKC. The '334 patent further discloses that the amount of inhibitor contemplated would not significantly inhibit other isozymes of PKC.
Thus, there remains a need in the art for unique compounds capable of selectively inhibiting active PKG in a peripheral nervous system. Inhibition of the active kinase would both prevent its transport from the periphery as well as block its activity in the cell body.